Gating strategy for the KIR panel. PBMC were stained according to the strategy outlined for NK staining panel A. A gating strategy was optimized to identify certain KIR+ populations where specific antibodies were not available. After gating on NK cells as described in the legend to Fig. 11.14.2–1, CD158b (which binds to both KIR2DL2 and KIR2DL3) was gated against KIR2DL3. Matched KIR genotyping data confirmed that individuals homozygous for KIR2DL2 displayed a CD158bpos KIR2DL3neg phenotype (A), individuals homozygous for KIR2DL3 displayed a CD158bpos KIR2DL3pos phenotype (B), and heterozygous individuals displayed populations of both CD158bpos KIR2DL3neg and CD158bpos KIR2DL3pos cells (C).

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Figure 11.14.2–2

Gating strategy for the KIR panel. PBMC were stained according to the strategy outlined for NK staining panel A. A gating strategy was optimized to identify certain KIR+ populations where specific antibodies were not available. After gating on NK cells as described in the legend to Fig. 11.14.2–1, CD158b (which binds to both KIR2DL2 and KIR2DL3) was gated against KIR2DL3. Matched KIR genotyping data confirmed that individuals homozygous for KIR2DL2 displayed a CD158bpos KIR2DL3neg phenotype (A), individuals homozygous for KIR2DL3 displayed a CD158bpos KIR2DL3pos phenotype (B), and heterozygous individuals displayed populations of both CD158bpos KIR2DL3neg and CD158bpos KIR2DL3pos cells (C).

NK cell degranulation assay. Following stimulation for 5 h with medium, K562, 721.221, or antibody-coated p815 cells in the presence of anti-CD107a antibody, brefeldin A, and monensin, PBMC were stained according to the degranulation antibody panel in Table 11.14.5–1. NK cells were identified as demonstrated in Fig. 11.14.2–1, and the expression of CD107a and intracellular cytokines was assessed on this population. Robust but variable responses were observed in response to all cell lines, and some background CD107a and MIP1β expression was seen, as expected. Gates for CD107a and MIP1β were set using FMOs, whereas gates for tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) were set based on the medium-only negative control.

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Figure 11.14.3–1

NK cell degranulation assay. Following stimulation for 5 h with medium, K562, 721.221, or antibody-coated p815 cells in the presence of anti-CD107a antibody, brefeldin A, and monensin, PBMC were stained according to the degranulation antibody panel in Table 11.14.5–1. NK cells were identified as demonstrated in Fig. 11.14.2–1, and the expression of CD107a and intracellular cytokines was assessed on this population. Robust but variable responses were observed in response to all cell lines, and some background CD107a and MIP1β expression was seen, as expected. Gates for CD107a and MIP1β were set using FMOs, whereas gates for tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) were set based on the medium-only negative control.

NK cell fluorescence killing assay. K562 target cells were stained with the membrane dye PKH26 and the cytoplasmic dye CFSE before being incubated for 5 h with either medium or PBMC effector cells at an effector/target ratio of 10:1. A separate tube of effectors only was also made. After gating on the targets cells (A), the effector-only sample was used to exclude the effector cell population (B), and the target cell-only sample was used to set the gate for viable cells and to measure background (C). Killed cells were identified as PKH26+ CFSElow, and the killing ability was reported as the percentage of targets displaying this phenotype (D).

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Figure 11.14.4–1

NK cell fluorescence killing assay. K562 target cells were stained with the membrane dye PKH26 and the cytoplasmic dye CFSE before being incubated for 5 h with either medium or PBMC effector cells at an effector/target ratio of 10:1. A separate tube of effectors only was also made. After gating on the targets cells (A), the effector-only sample was used to exclude the effector cell population (B), and the target cell-only sample was used to set the gate for viable cells and to measure background (C). Killed cells were identified as PKH26+ CFSElow, and the killing ability was reported as the percentage of targets displaying this phenotype (D).